Abstract

Recently, microarrays of synthetic long sense-oriented oligonucleotides were introduced as an alternative expression profiling platform with distinct advantages to both cDNA arrays and commercial arrays produced by in situ synthesis of multiple short oligonucleotides per gene. However, gene expression analysis using microarrays of long oligonucleotides is limited in that it requires substantial amounts of RNA. The objective of this thesis was to develop protocols that allow for the analysis of gene expression even in minimal samples. Two different approaches were taken, one that amplifies the RNA target material before hybridization and another that amplifies the signal generated on the array. Most existing target amplification protocols linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labeling by reverse transcription and hybridization to sense-oriented oligonucleotide arrays. Here, a novel protocol (TAcKLE) is introduced in which a combination of two reverse and one forward transcription reactions followed by dye-incorporation using the Klenow fragment of E. coli DNA polymerase I generates fluorescent antisense cDNA. This protocol provides high fidelity and up to 105-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples. Signal amplification is another option if only minimal amounts of sample material are available. Therefore, a method was evaluated that uses on-chip rolling circle replication of circularized oligonucleotides for the amplified detection of gene expression profiles. This principle should allow for a faster and cheaper experimental procedure, circumventing sequence-dependent amplification bias. The preliminary results provide evidence for the method’s applicability, but further experiments are required to reduce the required amount of starting material and to define a stable protocol. As the TAcKLE protocol performed particularly well, it was subsequently applied to evaluate the utility of spotted oligonucleotide microarrays compared to a widely-used and accepted commercial reference platform. There are numerous ways to perform global transcriptional profiling, among which microarray technology has certainly gained a premier position. The comparison of gene expression measurements obtained with different array-based approaches is therefore of substantial interest in order to clarify whether inter-platform differences may conceal biologically significant information. To address this concern, global gene expression was analyzed in a set of clinical head and neck squamous cell carcinoma samples, using both spotted oligonucleotide microarrays made from a large collection of 70-mer probes and commercial arrays produced by in situ synthesis of sets of multiple 25-mer oligonucleotides per gene. Expression measurements were compared for 4,425 genes represented on both platforms, which revealed strong correlations between the corresponding data sets and similar profiles of relative gene expression. In conclusion, combining the TAcKLE protocol with spotted oligonucleotide arrays is an attractive alternative for transcriptional profiling of limited source material, offering a high potential for gene expression analysis in a multitude of disease situations.